The evolutionarily conserved TSC/Rheb pathway activates Notch in tuberous sclerosis complex and Drosophila external sensory organ development

pathway activates Notch in tuberous sclerosis

complex and Drosophila external sensory organ

development

Magdalena Karbowniczek, … , Fabrice Roegiers, Elizabeth

Petri Henske

J Clin Invest.

2010;

120(1)

:93-102.

https://doi.org/10.1172/JCI40221

.

Mutations in either of the genes encoding the tuberous sclerosis complex (TSC),

TSC1

and

TSC2

, result in a multisystem tumor disorder characterized by lesions with unusual lineage

expression patterns. How these unusual cell-fate determination patterns are generated is

unclear. We therefore investigated the role of the TSC in the

Drosophila

external sensory

organ (ESO), a classic model of asymmetric cell division. In normal development, the

sensory organ precursor cell divides asymmetrically through differential regulation of Notch

signaling to produce a pIIa and a pIIb cell. We report here that inactivation of

Tsc1

and

overexpression of the Ras homolog Rheb each resulted in duplication of the bristle and

socket cells, progeny of the pIIa cell, and loss of the neuronal cell, a product of pIIb cell

division. Live imaging of ESO development revealed this cell-fate switch occurred at the

pIIa-pIIb 2-cell stage. In human angiomyolipomas, benign renal neoplasms often found in

tuberous sclerosis patients, we found evidence of Notch receptor cleavage and Notch target

gene activation. Further, an angiomyolipoma-derived cell line carrying biallelic TSC2

mutations exhibited TSC2- and Rheb-dependent Notch activation. Finally, inhibition of

Notch signaling using a

g

-secretase inhibitor suppressed proliferation of

Tsc2

-null rat cells

in a xenograft model. Together, these data indicate that the TSC and Rheb regulate

Notch-dependent cell-fate decision in

Drosophila

and Notch activity in mammalian cells […]

Research Article

Oncology

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Research article

The evolutionarily conserved TSC/Rheb

pathway activates Notch in tuberous sclerosis

complex and

Drosophila

external

sensory organ development

Magdalena Karbowniczek,1 _{Diana Zitserman,}1 _{Damir Khabibullin,}2 _{Tiffiney Hartman,}1

Jane Yu,2 _{Tasha Morrison,}2 _{Emmanuelle Nicolas,}1 _{Rachel Squillace,}3

Fabrice Roegiers,1 _{and Elizabeth Petri Henske}2

1_{Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA.} 2_{Division of Pulmonary and Critical Care Medicine, Brigham and}

Women’s Hospital, Boston, Massachusetts, USA. 3_{The Rothberg Institute for Childhood Diseases, Guilford, Connecticut, USA.}

Mutations in either of the genes encoding the tuberous sclerosis complex (TSC),

TSC1

and

TSC2

, result in a

multisystem tumor disorder characterized by lesions with unusual lineage expression patterns. How these

unusual cell-fate determination patterns are generated is unclear. We therefore investigated the role of the

TSC in the

Drosophila

external sensory organ (ESO), a classic model of asymmetric cell division. In normal

development, the sensory organ precursor cell divides asymmetrically through differential regulation of Notch

signaling to produce a pIIa and a pIIb cell. We report here that inactivation of

Tsc1

and overexpression of the

Ras homolog Rheb each resulted in duplication of the bristle and socket cells, progeny of the pIIa cell, and

loss of the neuronal cell, a product of pIIb cell division. Live imaging of ESO development revealed this

cell-fate switch occurred at the pIIa-pIIb 2-cell stage. In human angiomyolipomas, benign renal neoplasms often

found in tuberous sclerosis patients, we found evidence of Notch receptor cleavage and Notch target gene

activation. Further, an angiomyolipoma-derived cell line carrying biallelic TSC2 mutations exhibited TSC2-

and Rheb-dependent Notch activation. Finally, inhibition of Notch signaling using a

γ

-secretase inhibitor

suppressed proliferation of

Tsc2

-null rat cells in a xenograft model. Together, these data indicate that the TSC

and Rheb regulate Notch-dependent cell-fate decision in

Drosophila

and Notch activity in mammalian cells and

that Notch dysregulation may underlie some of the distinctive clinical and pathologic features of TSC.

Introduction

Tuberous sclerosis complex (TSC) is a multisystem tumor sup-pressor gene syndrome caused by mutations that inactivate either  TSC1 or TSC2. TSC is characterized by neurologic disease (sei- zures, mental retardation, and autism), pulmonary lymphangi-oleiomyomatosis (LAM), and hamartomatous tumors of the brain,  heart, kidney, and skin (1). The hamartin and tuberin proteins  (TSC1 and TSC2, respectively) function as a heterodimeric com- plex to inhibit the Ras homolog Rheb, which activates the mam-malian target of rapamycin (mTOR) complex 1 (TORC1) (2–7).  The TSC/Rheb/TOR pathway is conserved in Drosophila, in which  it has been shown to regulate cell size (8–15) and the timing of  neuronal development (16).

Unusual cell lineage expression patterns are a hallmark of TSC  lesions in humans. The central nervous system lesions in TSC  include cortical tubers and subependymal giant cell astrocytomas,  both of which contain distinctive “giant cells” that inappropriately  coexpress neuronal and glial-lineage markers (17, 18). Outside the  central nervous system, pulmonary LAM and renal angiomyoli-pomas are among the most clinically devastating features of TSC  (19). LAM cells and angiomyolipoma cells express both smooth  muscle and melanocytic markers, and angiomyolipomas exhibit  differentiation plasticity, with vascular, smooth muscle, and fat 

cells all arising from a common precursor cell (20). The molecular  mechanisms underlying these unusual cell-fate determination pat-terns in TSC tumors are not well understood.

To study the potential role of the TSC signaling network in cell-fate determination, we used the Drosophila external sensory organ  (ESO), which is a well-studied model of asymmetric cell division.  The sensory organ precursor (SOP) cell divides asymmetrically to  produce 2 daughter cells, the pIIa cell and the pIIb cell. The pIIa cell  divides once more to form the external cells of the ESO (the socket  and hair cells), and the pIIb cells divides twice to generate a neuron,  a sheath cell, and an apoptotic glial cell (Figure 1A). The asymmetry  between the pIIa and pIIb is established by differential regulation of  Notch signaling. Activation of Notch signaling in the pIIa cell and  suppression of Notch in the pIIb cell is controlled through multiple  mechanisms, including asymmetric segregation of the Notch antag-onist Numb to the pIIb cell during SOP mitosis (Figure 1A).

Here we show that inactivation of Tsc1 or overexpression of  Rheb in SOP cells results in a phenotype consistent with ectopic  Notch activation: duplication of the pIIa cell and its progeny, the  hair and socket cell, at the expense of the pIIb cell. We further find  evidence for Notch receptor cleavage and Notch target gene activa- tion in human angiomyolipoma tumors and an angiomyolipoma-derived cell line with biallelic TSC2 inactivation. Our results reveal  what we believe to be a novel, evolutionarily conserved pathway,  in which the TSC1/TSC2/Rheb proteins regulate the activity of 

Notch, which in turn may  act to regulate cell fate and cell prolif-Conflict of interest: The authors have declared that no conflict of interest exists.

Citation for this article: J. Clin. Invest. 120:93–102 (2010). doi:10.1172/JCI40221.

research article

eration. Notch activation may contribute to the unusual clinical  manifestations of TSC, and targeting the Notch signaling network  may provide a new clinical strategy for the therapy of TSC.

Results

Rheb regulates Notch-dependent cell-fate decisions in Drosophila. Based  on previous observations that central nervous system lesions in  TSC show inappropriate expression of neural and glial markers  (17, 18), we asked whether TSC loss or Rheb activation results in  cell-fate defects in the Drosophila ESO, a well-studied system for  understanding neural cell fate choice, based on Notch signal-ing activation (Figure 1A). We generated mosaic clones of 3 Tsc1  mutant alleles (Tsc1Q87X_, Tsc1W243X_{, and Tsc1}R453X

) on the fly tho-rax. Within these clones we observed 2 phenotypes: double bristles  (twinning) and patches of bald cuticle (balding) (Figure 1B). We  quantified these phenotypes in 17 flies with GFP-marked mosaic  clones of 2 of the alleles, W243X and R453X (Figure 1D), using  the mosaic analysis with a repressible cell marker (MARCM) sys-tem (see Methods). Twinning was present in 1.6% of the Tsc1W243X

ESOs and 5% of the Tsc1R453X

 ESOs (Supplemental Table 1; sup-plemental material available online with this article; doi:10.1172/ JCI40221DS1). Balding (lack of bristles) was observed in 33% of  the GFP-positive SOPs. To determine whether these cell fate phenotypes were a conse- quence of hyperactivation of Rheb, the target of the GTPase-acti- vating domain of tuberin (TSC2), we used the Gal4/upstream acti-vating sequence (Gal4/UAS) system (21) to express Rheb within  the SOP lineage, using the scabrous  promoter (21, 22). Three per-cent of Rheb-expressing ESOs showed bristle twinning (Figure 1,  B, E, and F, and Supplemental Table 1), similar to the Tsc mutant  flies. Duplication of external cell fates at the expense of internal  cells in the ESO lineage is a hallmark of inappropriate activation  of Notch signaling. We therefore examined whether the observed  increase in external cells (twinning) in Tsc and Rheb mutant cells  was accompanied by a loss of internal cells, using specific mark-ers for the neuronal and socket cells, embryonic lethal, abnormal  vision (ELAV) and suppressor of hairless [Su(H)], respectively. We  found that ESOs with twinning were devoid of ELAV-positive cells  (Figure 1, D and E), indicating lack of the neuron, and Su(H) was  expressed in 2 cells instead of 1 cell (Figure 1D), indicating dupli-cation of the socket cell. These data indicate that loss-of-function  of the TSC proteins causes Rheb-mediated defects in ESO develop-ment, with duplication of the external cells (the socket and hair)  and loss of the internal neuronal cell. In order to further probe the nature of the defects we observed in  Tsc mutant and Rheb overexpressing cells, we used live cell imaging  to monitor asymmetric division and differentiation of ESO cells  in living pupae of wild-type, Tsc mutant, and Rheb overexpress-ing cells. We used tau-GFP to observe the dynamics of the mitotic  spindle and partner of numb–GFP (Pon-GFP) as a reporter of cell  fate determinant asymmetric localization. Using this system, we  visualized the development of the sensory organ from the division  of the pI cell to morphogenesis of the differentiated sensory organ.  In both Tsc1 mutant and Rheb overexpressing cells, we observed  delays or premature arrest of the cell cycle and increased apoptosis  compared with wild-type cells (Supplemental Table 2 and Supple-mental Figure 1). Cell cycle arrest and apoptosis resulted in the  loss of sensory organs, consistent with the balding phenotype we  observe in adult flies (Supplemental Figure 1). We next focused on the analysis of asymmetric cell division in  sensory organs that displayed the twin bristle phenotype. In wild- type SOP cell division, Pon formed a distinct crescent at the ante-rior cortex of the pI cell. As the pI cell divided, this crescent was  segregated asymmetrically to the anterior pIIb daughter cell, as  shown previously (23, 24). Interestingly, in Rheb overexpressing  ESOs that subsequently developed duplication of hair and socket  cells, we observed asymmetric targeting of Pon-GFP to the anterior  cell cortex of the pI cell during mitosis and segregation of Pon-GFP to the pIIb daughter cell (Figure 1F). These data suggest that  the bristle duplication phenotype is not likely a result defective  segregation of cell fate determinants, such as Pon and Numb, dur-ing asymmetric cell division. While the pI cell appears to divide in the correct orientation along  the anterior-posterior axis under conditions of Rheb overexpres-sion, our live imaging revealed a striking change in mitotic spindle  alignment of the pIIb cells. In the wild-type fly, the pIIb cell aligns  its spindle along the apical-basal axis, while the pIIa cell spindle  aligns along the anterior-posterior axis (23, 25–27). In the Rheb-expressing flies, 12% of the pIIb cells aligned their spindles along  the anterior-posterior or medial-lateral axis, instead of the apical- basal axis. Importantly, the pIIb cells with misaligned spindles sub-sequently gave rise to duplicate bristle and socket cells (Figure 1F).  Despite the change in orientation of the mitotic spindle, Pon-GFP  was correctly segregated asymmetrically to only 1 daughter cell.  These findings are consistent with model whereby Rheb induces a  pIIb-to-pIIa cell-fate switch by promoting ectopic Notch activation,  resulting in the twinning phenotype (Figure 1, C–F).

Genetic interaction of Rheb with genes involved in sensory organ develop-ment. Our data indicate that Rheb activation or Tsc inactivation  results in incorrect cell fate specification in the pIIb cell, despite  correct asymmetric targeting of cell fate determinants such as  Figure 1

Numb during SOP mitosis. In order to determine whether Rheb  acts in the same pathway or in a parallel pathway to Numb, we used  a genetic approach and assessed the interaction between Rheb and  3 other genes that impact Notch activity: a nonphosphorylatable  form (3A) of lethal giant larvae [l(2)gl], nuclear fallout (Nuf), and  Rab11), nuclear fallout (Nuf), and Rab11. Nuf and Rab11 regulate  Notch activity by controlling Delta trafficking (28), while l(2)gl3A  regulates Notch activity by controlling the asymmetric targeting  of Numb (29). When combined with l(2)gl3A expression, Rheb  synergistically enhanced the twinning phenotype (Figure 2 and  Supplemental Table 1), while when combined with Rab11 or Nuf,  Rheb had only a minor impact on twinning (Figure 2A and Sup-plemental Table 1). From these data we conclude that Rheb likely  functions to control Notch signaling during ESO development via  a pathway that is parallel to asymmetric targeting of Numb.

Renal angiomyolipomas express Notch and Delta and have higher levels of Notch activity than normal kidney . We next explored the hypothe-sis that the TSC pathway regulates Notch activity in mammalian  cells. First, we asked whether angiomyolipomas express Notch  and Delta. Using real-time RT-PCR, we tested 4 angiomyolipomas  from a TSC patient and found that NOTCH1 and delta-like 1  

(DLL1) were expressed at levels similar to or higher than nor-mal kidney tissue from the same patient (Figure 3, A and B). The  Notch transcriptional target hairy and enhancer of split 1 (HES1)  was expressed in all 4 angiomyolipomas at levels higher than in  normal kidney, suggesting that Notch signaling is active in angi- omyolipomas (Figure 3C). To directly test whether Notch signal-ing is activated, we tested additional sporadic angiomyolipomas  by Western immunoblot using anti-Hes1 and an antibody to the  carboxy terminus of Notch. Notch protein levels and activation  of the Notch receptor, which was identified by examination of  the 110-kDa band representing the γ-secretase cleavage product  of Notch1, were enhanced in all 4 tumors compared with normal  kidney from the same patient. Furthermore, the Hes1 protein  level was higher in all 4 angiomyolipomas compared with that  in normal kidney (Figure 4). These data indicate that Notch is  active in angiomyolipomas.

TSC2 and Rheb regulate Notch signaling in an angiomyolipoma-derived cell line . Given this evidence that Notch is active in angiomyoli-poma tumors, we next investigated whether Notch activity is  Rheb dependent, using the 621-101 cell line, which was derived  from an angiomyolipoma and has inactivation of both alleles of  the TSC2 gene (30). Using real-time RT-PCR, we monitored the  levels of endogenous HES1 and hairy/enhancer-of-split related  with YRPW motif 1 (HEY1) in cells treated for 72 hours with the 

γ-secretase inhibitor, N-[N-(3,5-Difluorophenacetyl-l -alanyl)]-S- phenylglycine t-butyl ester (DAPT), which prevents Notch cleav-age and activation. DAPT treatment decreased HES1 transcript  level by 60% (Figure 5A), suggesting that these cells have Notch  activity. Next, to determine whether this Notch activity is Rheb  dependent, 621-101 cells were treated with Rheb siRNA. Deple-tion of Rheb significantly decreased HES1 and HEY1 transcript  levels (Figure 5B). To further confirm that Rheb regulates HES1  and HEY1 levels, we reexpressed TSC2, which decreases the GTP- bound active form of Rheb via tuberin’s GTPase-activating pro-Figure 2

Genetic interaction of Rheb with other genes involved in sensory organ development. (A) Quantification of the twinning phenotype in scaGal4, UAS-Rab11 (Rab11); scaGal4, UAS-Rheb/Rab11 (Rheb/Rab11); sca-Gal4-UAS-Nuf (Nuf); scaGal4, UAS-Rheb/Nuf (Rheb/Nuf); scaGal4, UAS-Rheb/CyO (Rheb); scaGal4, UAS-l(2)gl3A [l(2)gl3A]; and sca-Gal4, UAS-Rheb/l(2)gl3A [Rheb/l(2)gl3A] flies (also see Supplemental Table 1). The bars indicate percentage of transformed ESOs. Data represent mean ± SEM. *P < 0.001. (B) Examples of the twinning phe-notype (arrows) in scaGal4, UAS-l(2)gl3A and scaGal4, UAS-Rheb/ l(2)gl3A flies. Original magnification, ×100.

Figure 3

research article

tein domain. Expression of TSC2 decreased endogenous HES1  and HEY1 transcript levels (Figure 5C), similarly to Rheb siRNA.  To test directly whether Rheb regulates Notch activity, 621-101  cells were treated with control or Rheb siRNA, and the presence of  cleaved Notch was examined by Western immunoblotting. Deple-tion of Rheb decreased the level of the 110-kDa cleaved Notch  product detected with anti–C terminus Notch1 antibody in the  whole cell lysate (Figure 5D, left) and anti-cleaved Notch1 Val  1744 antibody in the nuclear fraction (Figure 5D, right).

TSC2 and Rheb regulate Notch signaling in mammalian cells.  To  determine whether TSC and Rheb regulate Notch activity in cells  that are not derived from TSC patients, we measured the endog-enous level of HES1 transcript in Tsc2-null mouse embryonic  fibroblasts (MEFs). Consistent with our results from angiomyo-lipoma-derived cells, we found that Tsc2-null MEFs have higher  levels of HES1 relative to littermate-derived control MEFs (Figure  6A). Next, we used HES1- and HEY1-luciferase reporter assays to  monitor Notch activity in 3 melanoma-derived cell lines. Down-regulation of Rheb with siRNA decreased HEY1-luciferase activity  in all 3 cell lines and decreased HES1-luciferase activity in 2 of  the lines (Figure 6B). In HEK293 cells, downregulation of TSC2  resulted in a 3-fold increase in HEY1-luciferase activity (Figure  6C), and downregulation of Rheb resulted in a 40% decrease in  HEY1-luciferase activity (Figure 6D).

Rheb-dependent Notch activation is insensitive to TORC1 inhibition.  The most well-studied target of the TSC/Rheb pathway is TORC1.  Interestingly, in HEK293 cells treated with TSC2 siRNA, the  TORC1 inhibitor rapamycin did not block the increased HEY1  luciferase activity (Figure 6E). To further explore whether Rheb-dependent Notch activation is rapamycin sensitive, we monitored  endogenous levels of cleaved Notch by Western immunoblot in  621-101 cells. Treatment with DAPT for 72 hours (as a positive  control) decreased levels of the 110-kDa band representing cleaved  Notch (Figure 7A), which was also confirmed with an antibody  detecting specifically cleaved Notch1 at Val 1744 (data not shown).  Treatment with rapamycin (20 nM) did not inhibit cleaved Notch  (Figure 7A), consistent with the results using the luciferase assay  in HEK293 cells. These data suggest that Rheb’s effects on Notch  activity are TORC1 independent, with the caveat that it is increas- ingly recognized that TORC1 is not completely blocked by rapa- mycin. Therefore, we treated 621-101 cells with Torin 1, an ATP-competitive mTOR kinase inhibitor (31). Similarly to rapamycin,  Torin 1 inhibited the phosphorylation of ribosomal protein S6  but did not inhibit Notch cleavage (Figure 7B), while as a control 

DAPT inhibited Notch cleavage but did not affect phosphory-lation of S6. Finally, as a third approach to inhibit TORC1, we  downregulated Raptor using siRNA. Similarly to rapamycin or  Torin 1 treatment, downregulation of Raptor did not affect Notch  cleavage but decreased phosphorylation of ribosomal protein S6  (Figure 6C). These results suggest that Rheb’s activation of Notch  is TORC1 independent.

Inhibition of Notch signaling inhibits the growth of Tsc2-null xenograft tumors. To determine whether Notch activity contributes to the  growth of Tsc2-null cells, we injected Tsc2-null ELT3 cells, which  were derived from the Eker rat model of TSC (32), bilaterally  into the flank region of SCID mice. When the tumors reached  250 mm3_{, the animals were treated with the γ-secretase inhibitor,}

DAPT. DAPT inhibited tumor growth by more than 50% relative  to vehicle control (Figure 8A) and was associated with a significant  decrease in Ki-67 positivity (Figure 8B). DAPT’s inhibition of Hes1  expression was confirmed by RT-PCR and immunoblot (Figure  8C). These results suggest that Notch activation drives prolifera-tion, thereby promoting growth of Tsc2-null tumors.

Discussion

TSC is a multisystem disease, in which many tumors and lesions,  including cerebral cortical tubers, subependymal giant cell astro-cytomas, angiomyolipomas, and LAM, display unusual cell-fate  determination patterns. While activation of Rheb and TORC1  is believed to drive the proliferative component of tumors carry- ing TSC1 or TSC2 mutations, little is known about the molecu-lar basis of these cell lineage abnormalities. To investigate the  cell fate abnormalities in TSC, we used the Drosophila ESO as a  model. The ESO develops over approximately 6 hours, during  the pupal stage, through a series of 4 asymmetric cell divisions.  We tested 3 different Tsc1 mutant alleles and found that all 3  disrupted cell fate assignments in the ESO, leading to a double  bristle phenotype. The double bristle resulted from a cell-fate  switch, in which the pIIb cell, which normally has low Notch  activity and generates the internal cells, acquired the character-istics of the pIIa cell, which normally has high Notch activity  and gives rise to the hair and socket cells. Similar double bristles  were observed in flies overexpressing Rheb, further confirming  the role of the TSC/Rheb signaling axis in ESO development.  The impact of the TSC/Rheb pathway on cell fate appears to be  lineage specific, since we did not observe any impact of Rheb  expression in the wing. This result may explain why evidence  of Rheb-induced Notch pathway activation was not observed  in previous studies of the Drosophila eye (16) and could also be  related to the cell-lineage specificity of TSC tumors.

Figure 4

Evidence of Notch activation in angiomyolipomas. The level of cleaved Notch1 and Hes1 measured by Western blot in sporadic angiomyo-lipomas and normal kidney from 4 patients. Activated Notch1 was identified by examination of the 110-kDa band, which represents the

Multiple lines of data indicate that the regulation of Notch  activity by the TSC proteins and Rheb is evolutionarily conserved  between flies and mammals. Evidence of Notch pathway activa-tion was present in angiomyolipoma tumor specimens. TSC2 and  Rheb were found to regulate Notch cleavage and Notch target gene  activation in an angiomyolipoma-derived cell line carrying bial-lelic TSC2 mutations. Evidence of Notch regulation by TSC2 and  Rheb was also observed in melanoma-derived cell lines, MEFs, and  HEK293 cells. Furthermore, inhibition of Notch signaling blocked  the growth of Tsc2 -null xenograft tumors, suggesting that this con-nection that we believe to be novel between TSC/Rheb and Notch  activity may be related to the proliferative aspects of TSC lesions  as well as their cell-fate abnormalities.

Treatment with the TORC1 inhibitor rapamycin, the TORC1  and TORC2 inhibitor Torin 1, or downregulation of Raptor, a key  component of TORC1 (33), did not inhibit Notch activation in  TSC2-deficient cells. This is consistent with the lack of a twinning  phenotype in flies expressing the Rheb target, dTOR kinase, in  the SOP lineage (data not shown). These data suggest that Rheb’s  regulation of Notch activity occurs via a TORC1-independent  mechanism. TORC1 is the most well-studied effector of Rheb, but  there are other indications that Rheb has rapamycin-insensitive 

targets (34–36). The identification of TORC1-insensitive pathways  regulated by Rheb and TSC2 is particularly important clinically  because TORC1 inhibitors have only partial clinical efficacy in  patients with TSC and LAM. In the first completed trial, rapamy-cin as a single agent resulted in an approximately 50% regression of  angiomyolipomas over 12 months of treatment, with nearly com-plete regrowth after discontinuation (37).

The Notch signaling pathway plays a central role in the regu-lation of neuronal progenitor cell differentiation (38, 39) and is  required to maintain stem/progenitor cells in an undifferentiated  status (40). Notch also impacts a broad range of cellular events  during normal development and in tumors, including prolifera-tion and migration (41–43). We speculate that Notch signaling in  TSC2-deficient progenitor cells may underlie some of the clinical  manifestations of TSC, by altering the terminal differentiation of  these cells with subsequent inappropriate differentiation and/or  proliferation. If this is correct, then a “pulse” of Notch inhibition  could promote the completion of differentiation and thereby  block further proliferation.

In summary, we report here that activation of Rheb leads to  abnormalities in asymmetric cell division in Drosophila SOP cells.  In mammalian cells, Rheb activates Notch, and Notch inhibition  Figure 5

research article

attenuates the growth of Tsc2-null cells in vivo. These findings  reveal what we believe to be a previously unrecognized, evolution-arily conserved regulatory function of the TSC and Rheb proteins.  Notch activation may contribute to the striking degree of cell-fate 

plasticity observed in TSC tumors, with angiomyolipomas dif-ferentiating into smooth muscle, fat, and vessels and expressing  melanocytic markers, as well as in cells within cortical tubers and  subependymal giant cell astrocytomas expressing both neuronal  and glial markers. Notch activation could also contribute to the  neurologic manifestations of TSC, which include seizures, mental  Figure 6

Rheb-dependent Notch activation in mammalian cells. (A) Endogenous HES1 transcript levels measured by real-time PCR were higher in

Tsc2–/–_p53–/– _{MEFs, compared with Tsc2}+/+_p53–/– _{MEFs. Data represent mean ± SEM. *P < 0.05. (B) Rheb siRNA decreased HEY1-luciferase}

retardation, and autism. Finally, our work may highlight elements  of the Notch signaling pathway (Notch, its ligands, and enzymes  required for signaling activation) as potential therapeutic targets  for TSC and LAM patients, for whom novel therapeutic concepts  are urgently needed.

Methods

Fly strains and statistics. To generate mosaic flies expressing mutant Tsc1 in  the SOP cells, we used the flippase recognition target/flippase recombina-tion enzyme (FLP/FRT) system. Mutant clones were identified using the 

MARCM system (44), which generates GFP-labeled cell clones with homo-zygous mutations. Yellow white (yw), ubx-FLP; scabrous-Gal4 (scaGal4),  UAS-Pon-GFP, UAS-tau-GFP; FRT82B tubulin-Gal 80 females were crossed  to yw; FRT82BTsc1Q87X_/TM3Sb–_{or to yw; FRT82BTsc1}W243X_/TM3Sb–_and

to yw; FRT82BTsc1R453X_/TM3Sb–

 males (provided by I. Hariharan, Univer-sity of California, Berkeley, California, USA).

To generate flies with transient expression of Rheb, we used the Gal4-UAS system (21). yw; scaGal4/curly of oyster (yw; scaGal4/CyO) females  were crossed to w; UAS Rheb males (provided by F. Tamanoi, University of  California, Los Angeles, California, USA).

Figure 7

Notch cleavage is insensitive to TORC1 inhibition in patient-derived cells. (A) Levels of the 110-kDa band representing the γ-secretase cleaved, active form of the Notch receptor, were decreased following treatment with the γ-secretase inhibitor DAPT as expected, while in rapamycin and DMSO treated cells, the 110-kDa band was present. Rapamycin inhibited the phosphorylation of ribosomal protein S6 as expected. (B) Treat-ment with the Tor kinase inhibitor Torin 1 did not inhibit Notch cleavage in 621-101 cells but did inhibit the phosphorylation of ribosomal protein S6. DAPT did inhibit the 110-kDa cleaved Notch band as expected. (C) Raptor downregulation did not inhibit Notch cleavage but did inhibit the phosphorylation of ribosomal protein S6. Actin and Creb were used as loading controls.

Figure 8

DAPT suppresses growth, proliferation, and Notch pathway activation in Tsc2 -null xenograft tumors. (A) Growth of

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To monitor the dynamics of SOP lineage division and spindle orienta-tion the scaGal4 promoter was used to drive UAS-Pon-GFP and tau-GFP  expression in Rheb transgenic flies. Female scaGal4, Pon-GFP, tau-GFP/

CyO flies (22, 23) were crossed to yw/w; scaGal4, UAS Rheb/CyO males. To test for enhancement of the twinning phenotype in Rheb flies, we  crossed females of either yw; scaGal4 or yw/w; scaGal4, UAS Rheb/CyO to  either males UAS-l(2)gl3A (provided by J. Knoblich, Institute of Molecular  Biotechnology of the Austrian Academy of Sciences, Vienna, Austria) or  UAS-Rab11-YFP (stock no. 9790) or UAS-EP3324 (Nuf) (both from Bloom-ington Drosophila Stock Center).

To statistically analyze the fly crosses for synergism, the sum of the  means of twinning in Rheb-expressing and l(2)gl3A-expressing flies was  compared with the mean of twinning in flies with Rheb and l(2)gl3A coex-pression. The chance that the observed results represented synergy rather  than additivity was calculated using the normal approximation to the dis-tributions of the 3 means values.

Time-lapse microscopy and image quantification. For time-lapse microscopy, 

Drosophila pupae were dissected and imaged as previously described (33).  For overnight movies, z stacks were acquired at 10-minute intervals using  the vertical section scan mode on a Bio-Rad 1024 confocal microscope.  Imaging was conducted over a large area; therefore, individual clusters are  pixilated. For imaging of fixed samples, z stacks were acquired by using  Nikon eclipse C1 confocal microscope.

RNA extraction and real-time RT-PCR. Total RNA was isolated using the  RNeasy kit (Qiagen) according to manufacturer’s instructions. RNA was  quantified using the Agilent 2100 BioAnalyzer in combination with a  RNA 6000 Nano LabChip. RNA was reverse-transcribed using Moloney  murine leukemia virus RT (Ambion) and a mixture of anchored oligo(dT)  and random decamers. For each sample, 2 RT reactions were performed  with inputs of 50 and 10 ng. Using TaqMan chemistry, 5′-nuclease assays  were run on a 7900HT Sequence Detection System (Applied Biosystems).  Ct values were converted to quantities (in arbitrary units) using a standard  curve (5 points; 5-fold dilutions) established with a calibrator sample. For  each sample, the 2 values of relative quantity were averaged. Antibodies. The following antibodies were used: rat anti-ELAV (1:300,  7E8A10; Developmental Studies Hybridoma Bank), rat anti-Su(H) (provid-ed by F. Schweisguth, École Normale Supérieure, Paris, France), anti-mouse  Alexa Fluor 555 (Invitrogen), mouse anti-tubulin and mouse anti-actin  (Sigma-Aldrich), and rabbit anti-Raptor, rabbit anti–phospho-S235/236  S6 ribosomal protein, anti-cleaved Notch1 Val 1744 (lot no. 5; Cell Sig-naling Technology), rabbit anti-Notch C terminus and rabbit anti-tuberin  (Santa Cruz Biotechnology Inc.), rabbit anti-Rheb and rabbit anti-HES1  (Abcam), and rabbit anti-Ki67 (Vector Laboratories Inc.).

siRNA and luciferase assays . For siRNA downregulation, cells were transfect-ed with 100 nM Rheb, TSC2, or Raptor SMARTpool siRNA, or nonspecific  control siRNA (all from Dharmacon) using Mirus Trans-IT TKO Transfec- tion reagent (Mirus). For luciferase assays, HEK293 cells were first trans- fected with the indicated siRNA and then, 24 hours later, with pHEY1-lucif-erase DNA or promoter-less luciferase control. Melanoma-derived cells were  transfected with pHEY1-luciferase or pHES1-luciferase DNA or promoter-less luciferase control simultaneously with siRNA. Both, pHEY1-luciferase  and pHES1-luciferase DNA constructs were provided by M. Gessler (Univer-sity of Wuerzburg, Wuerzburg, Germany). Luciferase levels were measured  after addition of luciferase substrate, and the difference of relative luciferase  units after correction to background (luciferase control) was calculated.

Nuclear fractionation. The preparation of nuclear extracts was performed  using CelLytic NuCLEAR Extraction Kit (Sigma-Aldrich). Briefly, cells were  allow swell with hypotonic buffer, then were disrupted using syringe and  needle. The cytoplasmic fraction was removed, and nuclear proteins were  released from the nuclei by a high salt buffer.

DAPT, rapamycin, and Torin 1 treatment. Cells were treated for indicated  times with 10 μM DAPT (Calbiochem), 20 nM rapamycin (Biomol), or 250  nM Torin 1 (provided by D. Sabatini, Whitehead Institute for Biomedical  Research, Cambridge, Massachusetts, USA and N. Gray, Dana Farber Can-cer Institute, Boston, Massachusetts, USA).

Induction of subcutaneous tumors in nude mice. ELT3 cells were inoculated  bilaterally into the posterior back region of 6-week-old immunodeficient  C.B17 SCID female mice. Tumor length, width, and depth were monitored  with a Vernier caliper. When tumors reached 250 mm3_{, mice were randomly}

assigned to intraperitoneal DAPT (Calbiochem) at a dose of 10 mg/kg/d  for 3 consecutive days, followed by 4 days without treatment (45) or vehicle  control. The Fox Chase Cancer Center Institutional Animal Care and Use  Committee approved this protocol.

Statistics. Both 2-tailed and 1-tailed Student’s t  tests were used for all statisti-cal analysis. P values of less than 0.05 were considered statistically significant.

Acknowledgments We thank Sandy Jablonski and the Fox Chase Cancer Center Con-focal Microscopy Facility for assistance with imaging; I. Hariharan,   J. Knoblich, F. Schweisguth, and F. Tamanoi for fly stocks and  reagents; M. Gessler for the HEY1-luciferase constructs; David Saba-tini and Nathanial Gray for Torin 1; and E. Golemis and J. Chernoff  for their critical review of the manuscript. This work was supported  by a postdoctoral fellowship to M. Karbowniczek from The LAM  Foundation (Cincinnati, Ohio, USA), the Tuberous Sclerosis Alliance  (Silver Spring, Maryland, USA), NIH RO1 DK51052 and HL81147  (to E.P. Henske), and NIH RO1 NS059971 (to F. Roegiers). Received for publication September 8, 2009, and accepted in  revised form October 21, 2009. Address correspondence to: Elizabeth Petri Henske, Brigham and  Women’s Hospital, One Blackfan Circle, Karp 6, Boston, Massa-chusetts 02115, USA. Phone: (617) 355-9049; Fax: (617) 355-9016;  E-mail: Ehenske@partners.org. Or to: Fabrice Roegiers, Fox Chase  Cancer Center, 333 Cottman Ave., Philadelphia, Pennsylvania  19111, USA. Phone: (215) 728-5518; Fax: (215) 214-2412; E-mail:  Fabrice.Roegiers@fccc.edu.   1. Crino PB, Henske EP. New developments in the  neurobiology of the tuberous sclerosis complex.  Neurology. 1999;53(7):1384–1390.   2. Plank TL, Yeung RS, Henske EP. Hamartin, the prod-uct of the tuberous sclerosis 1 (TSC1) gene, interacts  with tuberin and appears to be localized to cytoplas-mic vesicles. Cancer Res. 1998;58(21):4766–4770.  

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